MEFs are frozen down in a nitrogen freezer (located in the Hines lab). There, they can be stored for very long periods of time while maintaining viability. Cells can be stored in the -80 freezer for up to one week.

All reagents used to create MEF growth media should not be open outside a BSL-2 certified hood. Reserve a time for these hoods and follow all Aseptic Techniques outlined here on the wiki in previous protocols. All contamination in cell culture happens because of improper technique.

This procedure outlines how to properly freeze-down MEFs at a density of 5×10^6 cells/ml.

• MEF freeze-down media. (Growth media + 10% v/v DMSO)
• Cryovials
• Hank's buffered saline
• Trypsin
• 1ml micropipette and pipette tips
• -20 degree celsius freezer
• -80 degree celsius freezer

Note: * All cell culture work needs to be done in a BSL-2 certified hood, using proper aseptic technique.

1. Trypsinize and resuspend cells in Hank's buffered saline.
   • Aspirate off growth media from cells
   • Add 2 ml trypsin/EDTA
   • Incubate for 5 minutes in the incubator
   • Ensure cells are popped-off. Mechanically stimulate if necessary.
   • Add 3 ml growth media to the flask to deactivate trypsin.
   • Move cells to a centrifuge tube.
   • Centrifuge at 1500rpm for 5 minutes.
   • Aspirate off supernatant.
   • Resuspend sample in 1ml Hank's buffered saline.
2. Count cells using the cell counter in the cell culture facility using trypan blue and the reusable cell counter slide.
   • Take 10 ul cell suspension and place in a microcentrifuge tube.
   • Add 10 ul trypan blue stain to the microcentrifuge tube and mix carefully.
   • Using the reusable cell counter slide and a slide, add 10ul cell suspension and take to the cell counter Countess in the cell culture room. Record cell density.
   • Centrifuge cells at 1500rpm for 5 minutes.
   • During centrifugation, calculate the resuspension volume to get a cell suspension cell density of 5×10^6 cells per ml.
   • Label the cryovials with the cell type, date, initials, and MM number before starting procedure.
3. Resuspend cells in freeze-down media at a cell density of 5×10^6 cells per ml.
4. Pipette 1ml cell suspension into cryovials.
5. Move cryovials to the -20 degree freezer for 45 minutes.
6. Move cryovials to the -80 degree freezer overnight.
7. Move cryovials to the nitrogen freezer for long-term storage.

To Thaw MEFs

1. Prepare cell culture flask with growth media and place in incubator.
2. Retrieve frozen MEFs from nitrogen freezer.
3. Warm MEFs by submerging the lower part of the vial in water bath for 1 minute or until the only frozen part is the middle of the suspension.
4. Move to BSL2 hood.
5. Pipette up cell suspension and place into centrifuge tube with 4ml growth media.
6. Centrifuge at 1500RPM for 5 minutes.
7. Resuspend cells in growth media and plate into prepared cell culture flask. Label appropriately.